We explored the effect of
glucose-free
hypoxia/reoxygenation of cultured neonatal rat ventricular myocytes on
endothelin-1 and alpha 1-adrenoceptor induced activity of the
phosphoinositide cycle. At the same time the influence of these agonists on depletion of energy-rich
phosphates and cellular damage was assessed.
Glucose-free
hypoxia did not lead to an increase in basal
phospholipase C activity. However,
endothelin-1 (10(-8) M) and
phenylephrine (10(-5) M) evoked activation of
phospholipase C was attenuated after 60 min of
hypoxia and declined to 38% and 30% respectively of normoxic values after 90 min of
hypoxia. During
glucose-free
hypoxia,
phosphatidylinositol 4,5-bisphosphate, the substrate for
phospholipase C, but not
phosphatidylinositol or
phosphatidylinositol 4-monophosphate was seen to decline to 59% of normoxic values which was independent of activation of
phospholipase C by agonists.
ATP levels decreased after 30 min of
hypoxia and declined to 29% relative to normoxic control after 90 min of
hypoxia. Total
adenine nucleotide levels showed a similar pattern. The presence of 10(-8) M
endothelin-1 during
hypoxia did not influence the magnitude of
ATP depletion. However, after 15 min of reoxygenation, by itself not significantly leading to recovery of
ATP levels,
ATP levels were decreased by
endothelin-1 as compared to
hypoxia/reoxygenation without
phospholipase C agonist. Cellular damage as determined by
lactate dehydrogenase leakage was not observed during 90 min
hypoxia. Reoxygenation resulted in a three-fold increase in
enzyme release relative to normoxic control. In the presence of
endothelin-1 or
phenylephrine this reoxygenation-induced damage was respectively 1.7 and 3.0-fold increased. We conclude that the agonist-induced activity of the
phosphoinositide cycle is decreased in time during
glucose-free
hypoxia, partially through a decrease in
phosphatidylinositol 4,5-bisphosphate level. However, the remaining activity may give rise to increased cellular damage. As
endothelin-1 and alpha 1-adrenergic
amines are known to be released during
myocardial ischemia, stimulation of the
phosphoinositide cycle by these agonists might be an important factor in determining the magnitude of myocardial injury.